The ability of human immunodeficiency virus type 1 (HIV-1) to enter its target cell and establish an infection is dependent on interactions between functional HIV envelope glycoprotein (Env) complexes on the virus and receptors on the host cell. The HIV-1 Env complex is initially synthesized as the polyprotein precursor gp160, which undergoes oligomerization, disulfide bond formation and extensive glycosylation in the endoplasmic reticulum (Earl, Moss, and Doms, 1991) and is then proteolytically cleaved into the surface (gp120) and transmembrane (gp41) subunits by furin-like endo-proteases in the Golgi network (Fields, 1996; Hunter and Swanstrom, 1990). The resulting Env complex is a trimer, with three gp120 proteins associated non-covalently with three gp41 subunits.
During the entry process, gp120 interacts with the CD4 receptor, which triggers conformational changes that facilitate gp120 binding to a coreceptor, CCR5 or CXCR4 (Berger, Murphy, and Farber, 1999; Rizzuto et al., 1998). These interactions promote extensive conformational changes in the gp41 subunit that drive the insertion of the hydrophobic gp41 N-terminal region (fusion peptide) into the host cell membrane. Subsequently, formation of the six-helix bundle configuration of the three gp41 ectodomains forces the juxtaposition of the viral and cell membranes, promoting their fusion (Doms and Moore, 2000; Jones, Korte, and Blumenthal, 1998; Melikyan et al., 2000; Moore and Doms, 2003; Sattentau and Moore, 1991; Sullivan et al., 1998; Wu et al., 1996; Zhang et al., 1999).
The trimeric nature of the Env complex has been confirmed by various lines of evidence (Blacklow, Lu, and Kim, 1995; Center et al., 2002; Center et al., 2001; Chan et al., 1997; Chan and Kim, 1998; Lu, Blacklow, and Kim, 1995; Zhu et al., 2003), most recently by cryo-electron microscopy (Zanetti et al., 2006; Zhu et al., 2006). The trimer is held together by labile, non-covalent inter-subunit interactions. The weak interactions between gp120 and gp41, and between individual gp41 subunits, are probably necessary to permit the conformational changes that are necessary for the process of virus-cell fusion to proceed efficiently, but such instability of the Env complexes complicates the generation of soluble forms of Env trimers that are suitable for vaccine research and structural studies. To obtain soluble Env trimers, the transmembrane (TM) region and the cytoplasmic tail (CT) are routinely deleted from gp41 to create gp140 proteins that contain gp120 and the gp41 ectodomain (gp41ECTO). Attempts to stabilize the non-covalent inter-subunit interactions have included mutating the cleavage site within gp140 to make uncleaved oligomers (Chakrabarti et al., 2002; Srivastava et al., 2002; Yang et al., 2000; Yang et al., 2002; Zhang et al., 2001) and engineering of an inter-subunit disulfide bond (Binley et al., 2000) between gp120 and gp41 and an isoleucine to proline substitution at position 559 (I559P) in the N-terminal heptad region of gp41 ectodomain (SOSIP) (Sanders et al., 2002) to promote gp41-gp41 association.
Despite the efforts to stabilize the naturally unstable Env complexes, problems with the stability of the complexes still exist. For example, gp120 rapidly dissociates from gp41 when soluble forms of gp140 proteins are expressed, and trimeric gp140 proteins can degrade into dimers and monomers, or associate into tetramers (dimers of dimers) and aggregates (Earl et al., 1994; Schulke et al., 2002; Staropoli et al., 2000). Similarly, monomeric and oligomeric gp120-gp41 structures are found to be present on cells that express Env proteins, as are both gp41 stumps from which gp120 has been shed and uncleaved proteins that have evaded the host cell proteases that typically process gp160 (Herrera et al., 2005; Kuznetsov et al., 2003; Moore et al., 2006; Thomas et al., 1991; Wyatt and Sodroski, 1998; Zhu et al., 2003).
Thus, stable, Env-based vaccines that mimic the native trimer conformation of the native Env structure and that remain stable when used as immunogenns and vaccines are needed in the art to combat infection by HIV and its devastating consequences.